The formation of functional contacts between developing axons and their targets is an essential step in the establishment of neuronal circuits. At the neuromuscular junction (nmj), as at other chemical synapses, the number and distribution of neuro-transmitter receptors are critical factors in determining the response to presynaptic stimulation. The neuromuscular junction is the best understood chemical synapse. Most of what is known about chemical synapses in the brain was either first or most completely analyzed at the nerve-muscle synapse. The transmitter at the nmj, acetylcholine (ACh) was identified more than 50 years ago. The ACh Receptor (AChR) was the first receptor/ion channel to be purified. It is composed of four subunits encoded by four different genes.
A cardinal event in the formation of the NMJ is the accumulation of acetylcholine receptors (AChRs) in the muscle membrane opposed to the nerve terminal. At the mature junction, receptors are packed in the postsynaptic membrane at a density in excess of 20,000 receptors/sq.micrometer. The localization is striking in that more than 70% of the receptors are concentrated to the motor endplate, a region that comprises less than 0.1 percent of the muscle-surface membrane.
Before the arrival of the motor nerve, nicotinic AChRs are distributed relatively uniformly over the surface of muscle fibers. The distribution of receptors can be mapped physiologically by measuring the sensitivity of the muscle membrane with an intracellular recording electrode while applying ACh ionphoretically from an extracellular microelectrode filled with 1M ACh and placed at different points over the muscle surface. The distribution of receptors can also be visualized using radiolabeled or fluorochrome labeled .alpha.-bungarotoxin (BgTx), a snake venom protein that binds selectively and almost irreversibly to nicotinic AChR (the type of AChR in skeletal muscle), or with monoclonal antibodies directed against extracellular regions of the receptor.
These labeling techniques reveal a dramatic change in the distribution of AChRs after innervation of the muscle fiber. There is a large increase in the density of receptors at the site of innervation and a decrease in the density of receptors at extrasynaptic sites. AChRs begin to accumulate at developing junctions within a few hours after nerve-muscle contact and the onset of synaptic transmission This phenomenon has been studied extensively in cell cultures containing embryonic motor neurons and myotubes. Individual synaptic partners can be visualized directly and monitored over periods of time that extend from seconds to several days.
Although a few AChRs and AChR clusters are present on uninnervated embryonic myotubes and myoblasts, it is clear that ingrowing motor nerves induce new receptor clusters rather than seeking out pre-existing ones (Anderson et al. 1977 J. Physiol. 268: 757; Frank and Fischbach 1979 J. Cell Biol. 83: 142). At least two processes contribute to the accumulation of AChRs at developing synaptic junctions. First, motor neurons may promote the aggregation of receptors that were present on the myocycte before nerve-muscle contact. These receptors may diffuse within the plane of the membrane and become immobilized at the synaptic site, presumably by binding to sites within the cytoskeleton and/or extracellular matrix. Second, motor neurons may induce the target muscle to increase the synthesis and insertion of new receptors in the immediate vicinity of the synapse. At chick synaptic junctions, the majority of AChRs at newly formed synapses or neurite associated receptor patches (NARPs) are newly inserted (Role et al., 1985 J. Neurosci 5:2197).
The motor nerve terminal triggers other changes in the properties of the postsynaptic receptor. For instance, AChRs at junctional sites lose their ability to diffuse in the plane of the membrane and gradually become fixed at the site of the synapse. Additionally, AChRs at junctional sites have a much longer half-life than extrajunctional receptors. AChRs found at newly formed end-plates in embryonic chicks have a half-life of about 24 hours, which is similar to that of extracellular receptors. With increasing time after synapse formation, junctional receptors become more stable, turning over with a half-life of more than 120 hours, whereas extrajunctional receptors are not stabilized.
The motor nerve also induces a change in the functional properties of nicotinic AChRs after skeletal muscle is innervated. AChR channels in embryonic rat muscle have a relatively small conductance (about 30pS) but remain open for long periods (about 5-10 mS) and have therefore been termed slow channels. In contrast, junctional receptors at mature end-plates have a significantly larger conductance (about 50pS) but remain open for a much shorter period (usually only about 1 mS) and are called fast channels.
AChRs at mature mammalian neuromuscular junctions are pentameric protein complexes composed of four subunits in the ratio of .alpha..sub.2 .beta..epsilon..delta. (Mishina et al. 1986 Nature 321: 406; Gu et al. 1988 Neuron 1:117, incorporated by reference herein). Most, if not all, embryonic AChRs contain a different subunit, termed ".gamma.", in place of the .epsilon. subunit. When mixtures of .alpha., .beta., .delta., and .gamma. subunit mRNAs are injected into Xenopus oocytes, the expressed channels have the properties of embryonic receptors. When transcripts encoding the .epsilon.-subunit are substituted for the .gamma. subunit, the resulting channels have the properties of adult receptors. It is likely that this change in subunit composition, which occurs during the first 2 weeks after birth and is due to a switch in gene expression, accounts for the switch in properties of ACh-activated channels from slow channels to fast channels which occurs over approximately the same time course.
The influence of the nerve on the AChR distribution appears to be mediated at least in part by difusable factors released by the presynaptic nerve terminal. For instance, myotubes located close to a spinal cord explant have been shown to be more sensitive to iontophoretically applied ACh and bind more .sup.125 I-BgTx than do myotubes located some distance away (Cohen and Fischbach, 1977 Devel. Biol. 59:24). Aceytlcholine itself does not seem to be the molecule responsible for the clustering of AChRs, as evidenced by the lack of AChR clustering in response to local application of ACh, and the observation that receptor clustering can occur when all AChRs are blocked by drugs such as curare.
Progress has been made in identifying a putative trophic factor that can increase the rate of receptor insertion, and that can promote the transition from embryonic to adult-type nicotinic AChRs. An Acetylcholine Receptor-Inducing Activity (ARIA) has been partially purified from adult chicken brains (Jessell et al., 1979 PNAS 76: 5397; Buc-Caron et al., 1983 Div. Biol. 95: 378; Usdin and Fischbach 1986 J. Cell Biol 103: 493). The purification was based on a sensitive assay in which the initial rate of appearance of new surface membrane AChRs are measured with .sup.125 I-BgTx four hours after blocking all exposed (old) receptors with unlabeled BgTx (Devreotes and Farobrough, 1975 J. Cell Biol 65: 335). The pitied protein was shown to increase the rate of AChR synthesis several fold with a K.sub.app in the picomolar range. ARIA does not appear to increase total protein synthesis or alter the degradation of surface receptors, but has been shown to affect the levels of certain AChR subunit mRNAs (Harris et al., 1988 PNAS 85: 7669).
This activity was shown to co-migrate with a protein that migrates as a broad band centered at an apparent MW of 42 kd by SDS-PAGE (Usdin et al. 1986 J. Cell Biol. 103:493). A chicken prion-like protein (Ch-PrLP) emerged as a major protein and apparently the only sequenceable protein in preparations of this activity (Falls et al. (1990) Cold Spring Harbor Symp. Quant. Biol. 55: 397). Based on N-terminal amino acid sequence analysis, oligonucleotides, were generated having sequences corresponding to portions of the chemically determined sequence of the protein present in the SDS-polyacrylamide band in which the activity was present, and were used to isolate a cDNA from an embryonic chick cDNA library. The isolated cDNA encodes a chicken protein that is homologous to the mammalian prion protein (PrPc). This chicken prion-like protein (ch-PrLP) was shown to be identical to the mouse PrP at 33% of its amino acid positions, and appeared to contain similar structural domains (Harris et al. 1991 PNAS 88: 7664, incorporated by reference herein).. However, the Ch-PrLP was not active when expressed, and anti-Ch-PrLP antibodies do not precipitate receptor-inducing activity.